Assays and peptide substrate for determining aggrecan degrading metallo protease activity

ABSTRACT

This invention is directed to assays to determine the presence or absence of proteins that exhibit aggrecanase or ADMP activity. This invention also relates to peptides that acts as a substrates for ADMPs, their use in various assays to determine the presence or absence of ADMP activity, and their use as inhibitors of ADMP activity.

This application is a divisional application of U.S. application Ser.No. 09/975,813, filed on Oct. 12, 2001, which is a divisionalapplication of U.S. application Ser. No. 09/122,127, filed on Jul. 25,1997 (now U.S. Pat. No. 6,326,162), which claims benefit to three U.S.Provisional Applications: U.S. Provisional Application No. 60/053850filed on Jul. 25, 1997, U.S. Provisional Application No. 60/055836 filedon Aug. 15, 1997; and U.S. Provisional Application No. 60/062,169, filedon Oct. 16, 1997. Each of the priority applications is incorporated byreference in their entireties.

FIELD OF INVENTION

This invention is directed to various assays for determining aggrecanaseor aggrecan degrading metallo protease (ADMP) activity. This inventionalso relates to a peptide substrate for ADMPs; the use of the peptidesubstrate in various assays to determine the presence or absence of ADMPactivity; and the use of the substrate as an inhibitor of ADMP activity.

BACKGROUND OF THE INVENTION

Aggrecan is the major proteoglycan of cartilage and provides this tissuewith its mechanical properties of compressibility and elasticity. Inarthritic conditions one of the earliest changes observed in cartilagemorphology is the depletion of aggrecan, which appears to be due to anincreased rate of degradation. Mankin et al., (1970) J. Bone Joint Surg.52A, 424-434.

The aggrecan molecule comprises two N-terminal globular domains: G1 andG2. The G1 and G2 domains are separated by an approximately 150 residueinterglobular domain (IGD), followed by a long central glycosaminoglycan(GAG) attachment region and a C-terminal globular domain, G3. Hardinghamet al., (1992) in Articular Cartilage and Osteoarthritis: Aggrecan, TheChondroitin Sulfate/Keratan Sulfate Proteoglycan from Cartilage(Kuettner et al.) pp. 5-20, Raven Press, New York and Paulson et al.,(1987) Biochem. J. 245, 763-772. These aggrecan molecules interactthrough the G1 domain with hyaluronic acid and a link protein, to formlarge molecular weight aggregates which are trapped within the cartilagematrix. Hardingham et al., (1972) Biochim. Biophys. Acta 279, 401-405;Heinegard et al., (1974) J. Biol. Chem. 249, 4250-4256; and Hardingham,T. E. (1979) Biochem. J. 177, 237-247. Loss of aggrecan from cartilagein arthritic conditions involves proteolytic cleavage of the aggrecancore protein within the IGD, producing an N-terminal G-1 fragment boundto hyaluronic acid and the link protein within the matrix. A largeC-terminal GAG-containing aggrecan fragment is released and diffuses outof the cartilage matrix. Loss of the C-terminal fragment results incartilage having deficient mechanical properties. The deficiency arisesbecause the GAGs, which are present on the C-terminal portion of theaggrecan core protein, comprise components of aggrecan that impartbeneficial mechanical properties to the molecule through their highnegative charge and water binding capacity.

IGD has two major proteolytic cleavage sites: one between amino acidresidues Asn³⁴¹-Phe³⁴² and the other between residues Glu³⁷³-Ala³⁷⁴(human sequence enumeration). G1 fragments formed as a result ofcleavage at the Asn³⁴¹-Phe³⁴² site and at the Glu³⁷³-Ala³⁷⁴ site havebeen identified within articular cartilage. However, the “ARGSVIL”, isthe only N-terminal sequence identified on GAG-containing aggrecanC-terminal aggrecan fragments in synovial fluids of patients withosteoarthritis, inflammatory joint disease, and in the media fromcartilage explant and chondrocyte cultures stimulated with interleukin-1or retinoic acid. This finding suggests that the fragments were formedby cleavage between amino acid residues Glu³⁷³-Ala.³⁷⁴ Theseobservations suggest that cleavage at this site may be responsible forcartilage degradation. See Flannery et al., (1992) J. Biol. Chem. 267,1008-1014; Sandy et al., (1992) J. Clin. Invest. 69, 1512-1516;Lohmander et al., (1993) Arthritis Rheum. 36, 1214-1222; Sandy et al.,(1991) J. Biol. Chem. 266, 8198; Sandy et al., (1991) J. Biol. Chem.266, 8683-8685; Leulakis et al., (1992) Biochem. J. 264, 589-593; Ilicet al., (1992) Arch. Biochem. Biophys. 294, 115-122; and Lark et al.,(1995) J. Biol. Chem. 270, 2550-2556.

Although many matrix metalloproteases (e.g., MMP-1, -2, -3, -7, -8,-9and 13) have been shown to cleave aggrecan at the Asn³⁴¹-Phe³⁴² site invitro, digestion of aggrecan with a number of purified proteasesdemonstrates that there is no cleavage at the Glu³⁷³-Ala³⁷⁴ site. Fosanget al., (1992) J. Biol. Chem. 267, 19470-19474; Flannery et al., (1992)J. Biol. Chem. 267, 1008-1014; Fosang et al., (1993) Biochem. J. 295,273-276; Fosang et al., (1996) FEBS Lett. 380, 17-20; Flannery et al.,(1993) Orthop. Trans., 17, 677; and Fosang et al., (1994) Biochem. J.305, 347-351. Cleavage at this site has been attributed to a novel,proteolytic activity termed “aggrecanase” activity.

In addition to the Glu³⁷³-Ala³⁷⁴ bond within the IGD of aggrecan, fourpotential aggrecanase-sensitive sites have been identified within theC-terminus of the aggrecan core protein outside of the IGD. Loulakis etal., (1992) Biochem. J. 264, 589-593. and Sandy et al., (1995) ActaOrhtop Scand (Suppl 266) 66, 26-32. Although cleavage at these sitesoutside of the IGD would not be expected to release the major portion ofthe aggrecan molecule from the matrix, the cleavage sites maynonetheless be involved in processing of aggrecan within the matrix.

It follows from the foregoing considerations that a sensitive andspecific assay for detection of aggrecanase activity by ADMPs would bebeneficial to identify inhibitors of members of the ADMP family, whichcould serve as potential therapeutic agents for treatingaggrecanase-related disorders.

SUMMARY OF THE INVENTION

A preferred embodiment of the invention provides an assay thatfacilitates determination of ADMP activity.

A preferred embodiment of the invention provides an assay using purifiednative aggrecan or recombinant aggrecan as the substrate and monitoringproduct generation via an enzyme-linked immunosorbent assay (ELISA)using neoepitope antibodies to detect the newly formed N- or C-terminalaggrecan epitopes formed by specific cleavage at an ADMP-sensitive sitein the aggrecan core protein.

A preferred embodiment of the invention provides peptides that have beenfound to act as substrates for the family of ADMPs. One peptide, basedon the human aggrecan sequence around the Glu³⁷³-Ala³⁷⁴ ADMP-sensitivesite has the sequence: (SEQ ID NO:1)QTVTWPDMELPLPRNITEGE-ARGSVILTVKPIFEVSPSPL

A second peptide, based on the bovine aggrecan sequence around theGlu³⁷³-Ala³⁷⁴ ADMP-sensitive site has the sequence: (SEQ ID NO:2)QTVTWPDVELPLPRNITEGE-ARGSVILTAKPDFEVSPTAPE

Both peptides are capable of being cleaved at this specific recognitionsite by members of the family of ADMP proteins. A third peptide, basedon the human aggrecan sequence around the Ala¹⁷¹⁴-Gly¹⁷¹⁵ ADMP-sensitivesite has the sequence: (SEQ ID NO:3)ITFVDTSLVEVTPTTFKEEE-GLGSVELSGLPSGELGVSGTand is capable of being cleaved at this specific recognition site by thefamily of ADMP proteins.

A preferred embodiment of the invention provides assay formats andmethods of utilizing these peptide substrates for the detection andquantification of ADMP activity.

Another preferred embodiment of the invention provides a modifiedversion of the peptide substrates and a method for their use as aninhibitor of ADMP activity.

BRIEF DESCRIPTION OF THE DRAWINGS

Preferred embodiments of the invention have been chosen for purposes ofillustration and description, but are not intended in any way torestrict the scope of the invention. The preferred embodiments ofcertain aspects of the invention are shown in the accompanying drawingsdescribed below.

FIG. 1. Shows the activity of a biotinylated form of the 41-mer peptidesubstrate (41-PS) against ADMP enzymatic activity using the microplateassay format and the inhibition of that activity by a hydroxymateinhibitor compound.

FIG. 2. Shows the activity of a biotinylated form of the 41-mer peptidesubstrate (41-PS) against ADMP enzymatic activity using the HPLC assayformat.

FIG. 3. Shows the activity of the biotinylated form of the 41-merpeptide substrate (41-PS) against ADMP enzymatic activity using themicroplate assay format and the inhibition of that activity by the30-mer inhibitor peptide (30-IP),QTVTWP-DMELPLPRNITEGQARGSVILTVK-Biotin, the sequence of which is basedupon the sequence of the 41-PS.

DETAILED DESCRIPTION OF THE INVENTION

A family of ADMP proteins cleave the aggrecan core protein at theGlu³⁷³-Ala³⁷⁴ peptide bond and exhibit enzymatic activity referred to as“aggrecanase” activity. Flannery et al., (1992) J. Biol. Chem. 267,1008-1014. The presence of ADMP enzymatic activity can be determined bymonitoring the production of aggrecan fragments generated exclusively bycleavage at the Glu³⁷³-Ala³⁷⁴ peptide bond within the aggrecan coreprotein. These aggrecan fragments are detected using neoepitopeantibodies to the N- or C-terminal epitopes produced by specificcleavage at this ADMP-sensitive site. The neoepitope antibodies that maybe used include, but are not limited to, BC-3 monoclonal antibodies. See(Hughes, C. E., et al., Biochem. J. 306:799-804, 1995; U.S. ProvisionalPatent Application Ser. No. 60/006,684 and U.S. patent application Ser.No. 08/743,439.

ADMP activity may be detected by monitoring the production of fragmentsformed by cleavage at alternative ADMP-sensitive sites using neoepitopeantibodies to the C-or N-terminal epitopes generated by ADMP-specificcleavage at these sites. Alternative sites in the aggrecan core proteinencompass, but are not limited to, the E¹⁵⁴⁵-G¹⁵⁴⁶, E¹⁷¹⁴-G¹⁷¹⁵,E¹⁸¹⁹-A¹⁸²⁰, or E¹⁹¹⁹-L¹⁹²⁰ bond (numbering based on the human aggrecancore protein sequence).

A preferred assay format involves using purified native aggrecan orrecombinant aggrecan as the substrate with product detection via ELISA,herein referred to as the “Problot assay”, using neoepitope antibodiesto the C- or N-terminal epitopes generated upon specific cleavage atADMP-sensitive sites within the aggrecan core protein. Alternative sitesin the aggrecan core protein encompass, but are not limited to, theE¹⁵⁴⁵-G¹⁵⁴⁶, E¹⁷¹⁴-G¹⁷¹⁵, E¹⁸¹⁹-A¹⁸²⁰, or E¹⁹¹⁹-L¹⁹²⁰ bond (numberingbased on the human aggrecan core protein sequence). These human aggrecanADMP-sensitive cleavage sites are conserved in aggrecan from variousanimal species although the absolute numbering based on the sequence ofthe aggrecan core protein may vary from species to species. Conservedamino acid sequences in various species around conserved ADMP-sensitivesites are shown below. Human NITEGE³⁷³ ³⁷⁴ARGSVILT Bovine NITEGEARGSVILT Rat NITEGE ARGNVILT Mouse NVTEGE ALGSVILT Pig NITEGE ARGTVILTSheep NITEGE ARGNVILT Chicken NVTEEE ARGSI Horse NITEGE ARGNVILT HumanASTASELE¹⁵⁴⁵ ¹⁵⁴⁶GRGTIGIS Bovine ATTAGELE GRGTIDIS Mouse ATTSSELEGRGTIGIS Rat ATTASELE GRGTISVS Human PTTFKEEE¹⁷¹⁴ ¹⁷¹⁵GLGSVELS BovinePTTFKEEE GLGSVELS Rat PTTFREEE GLGSVELS Mouse PTTFREEE GLGSVELS HumanTQAPTAQE¹⁸¹⁹ ¹⁸²⁰AGEGPSGI Bovine TQAPTAQE AGEGPSGI Rat TLAPTAQE AGEGPSSIMouse TQAPTAQE AGEGPSGI Chicken TQTSVAQE VGEGPSGM Human TEPTISQE¹⁹¹⁹¹⁹²⁰LGQRPPVT Bovine TEPTVSQE LGQRPPVT Rat TEPTVSQE LGHGPSMT MouseTEPTVSQE LGHGPSMT Chicken TRPTVSQE LGGETAVT Dog TEPTVSQE LAQRPPVT

Thus, aggrecan from various animal species, including but not limitedto, bovine, dog, pig, rat, mouse, sheep, horse and chicken may also beused as a substrate for detecting ADMP activity.

The direct ELISA assay employs 96-well filtration plates containingpolyvinyl-denedifluoride (PVDF) cationically charged membranes. Thesemembranes are semi-selective in binding the highly negatively-chargedaggrecan, which allows for binding of detectable levels of neoepitopeantibody-reactive aggrecan fragments from solutions containing highlevels of other proteins.

Utilizing neoepitope antibodies allows detection of fragments formedspecifically by ADMP-mediated cleavage even in the presence of otherproteolytic activities which may be present in crude preparations. Thus,the Problot assay can be used to monitor ADMP activity in culture mediumcontaining other proteases, as well as to monitor the activity of thepurified ADMP enzyme. Therefore, this assay has particular use infollowing ADMP activity during purification from tissue or mediasamples, as well as for use in enzymatic assays to evaluate inhibitorsof the ADMP enzyme. The Problot assay can also be used to detectADMP-generated aggrecan fragments in culture media from tissue or cellcultures stimulated to induce ADMP-mediated degradation. This assay mayalso be useful for detecting ADMP-generated aggrecan fragments incartilage, synovial fluid, serum, urine or other biological samples frompatients with ADMP-associated diseases.

Peptide substrates are commonly employed in a variety of assays todetermine the presence of enzymes that catalyze the hydrolysis ofproteins. One skilled in the art would rely on the use of peptidesubstrates that are relatively short in length, generally consisting ofapproximately six to ten amino acids in length. These peptide substratestypically encompass amino acid sequences that bracket the knownhydrolysis site of the natural protein substrates. These peptidesubstrates, including those for matrix metalloproteases, serineproteases, aspartyl proteases, and aminopeptidases, are readilyavailable for use in a variety of enzymatic assays.

Further provided are peptides that have been found to act as substratesfor ADMPs. It is commonly known that short peptide sequences whichcontain the proper substrate cleavage site are quite acceptablesubstrates for many proteases. Copeland, R. A., Enzymes: A PracticalIntroduction to Structure, Mechanism and Data Analysis, VCH/Wiley, N.Y.,1996. However, no such peptide, even those containing as many as twentyamino acids, has been determined that will act as a suitable substratefor ADMPs. The peptides of the instant invention are unique in that itwas unexpectedly found that these longer, forty amino acid sequenceacted as very good substrates for ADMPs. One such peptide provided bythe invention, of the sequence: (SEQ ID NO:1)QTVTWPDMELPLPRNITEGE-ARGSVILTVKPIFEVSPSPL

comprises a 40 amino acid segment of the human aggrecan protein thatcontains the ITEGE³⁷³-³⁷⁴ARGS cleavage site present in aggrecan, and iscapable of being cleaved at this specific recognition site by the ADMPs.Since the human aggrecan ADMP-sensitive cleavage sites are conserved inaggrecan from various animal species, peptides based on the amino acidsequence around the ADMP-sensitive cleavage sites from other species canalso serve as substrates for ADMPs. A peptide substrate, similar to SEQID NO: 1, based on a 41 amino acid segment of the bovine aggrecanprotein, of the sequence (SEQ ID NO:2)QTVTWPDVELPLPRNITEGE-ARGSVILTAKPDFEVSPTAPEcontaining the E³⁷³-A³⁷⁴ cleavage site is also capable of being cleavedat this specific recognition site by the ADMPS.

Cleavage products are easily detected by using neoepitope antibodies tothe N-terminal or C-terminal fragments produced by specific cleavage atthe E³⁷³-A³⁷⁴ bond. Such antibodies include the monoclonal antibodyBC-3. Hughes, C. E., et al., Biochem. J. 306:799-804, 1995. The BC-3antibody recognizes the N-terminal epitope comprising the sequence“ARGS” which is the amino terminal portion of one of the productpeptides resulting from the ADMP activity of the enzyme.

One skilled in the art could readily design peptides of similar sizeencompassing the alternative ADMP-sensitive cleavage sites in theaggrecan core protein, encompassing, but not limited to, regions of themolecule containing the E¹⁵⁴⁵-G¹⁵⁴⁶, E¹⁷¹⁴-G¹⁷¹⁵, E¹⁸¹⁹-A¹⁸²⁰, orE¹⁹¹⁹-L¹⁹²⁰ bond (numbering based on the human aggrecan core proteinsequence). One such peptide provided by the invention, of the sequence:(SEQ ID NO:3) ITFVDTSLVEVTPTTFKEEE-GLGSVELSGLPSGELGVSGTcomprises a 40 amino acid segment of the human aggrecan protein thatcontains the KEEE¹⁷⁴-¹⁷¹⁵GLGS cleavage site present in the naturalprotein substrate, aggrecan, and is capable of being cleaved at thisspecific recognition site by the ADMPs.

A preferred form of the peptide substrate is a peptide that isbiotinylated at the carboxy terminus or amino terminus. Severalstreptavidin coated supports may be used. These include, but are notlimited to microplates, metallic and non-metallic beads, and membranes.

Another preferred assay format involves the direct analysis, byhigh-performance liquid chromatography (HPLC), of the cleavage fragmentsfrom the substrate that are generated by ADMP activity.

Another preferred embodiment of the invention is a substrate thatinhibits ADMP activity. It was discovered that esterification of the P1glutamic acid residue (GLU³⁷³) of the substrate peptide of SEQ ID NO: 1,or its replacement by glutamine abolish catalytic hydrolysis.Unexpectedly, the peptide containing the GLU to GLN substitution atamino acid position 373 (the P1-glutamine containing peptide) was shownto be a competitive inhibitor of the enzyme. Thus, a carboxylate residueat position P1 of the substrate appears to be involved in turnover byADMPs, but exerts less influence over initial substrate binding to theenzyme. This feature can be readily exploited by one trained in the artto design specific peptide and non-peptide inhibitors of this enzyme.

DEFINITIONS

As used herein, the following terms and expressions have the indicatedmeanings.

The term “aggrecan degrading metallo protease” (“ADMP”) activity asreferred to herein, refers to the enzymatic activity of a family ofpolypeptides which specifically cleave the protein aggrecan within theinterglobular domain at the Glu³⁷³-Ala³⁷⁴ peptide bond, but do notreadily cleave at the Asn341-Phe342 bond which is preferentially cleavedby matrix metalloproteases.

The term “amino acid” as used herein means an organic compoundcontaining both a basic amino group and an acidic carboxyl group.

The term “amino acid residue” as used herein means that portion of anamino acid (as defined herein) that is present in a peptide.

The term “peptide” as used herein means a compound that consists of twoor more amino acids (as defined herein) that are linked by means of apeptide bond. The term “peptide” also includes compounds containing bothpeptide and non-peptide components, such as pseudopeptide or peptidemimetic residues or other non-amino acid components. Such a compoundcontaining both peptide and non-peptide components may also be referredto as a “peptide analog”.

The term “peptide bond” means a covalent amide linkage formed by loss ofa molecule of water between the carboxyl group of one amino acid and theamino group of a second amino acid.

The term “substrate” refers to a molecule that is bound by the activesite and acted upon by the enzyme.

The term “solid-phase peptide synthesis” refers to the direct chemicalsynthesis of peptides utilizing an insoluble polymeric support as theanchor for the growing peptide, which is built up one amino acid at atime using a standard set of reactions in a repeating cycle. Merrifield,R. B., Science 232, 341-347 1986.

As used herein, the term “TMB” refers to 3,3′,5,5′-tetramethylbenzidine.

The term “neoepitope antibody” refers to an antibody which specificallyrecognizes a new N-terminal amino acid sequence or new C-terminal aminoacid sequence generated by proteolytic cleavage but does not recognizethese same sequences of amino acids when they are present within theintact protein.

As used herein, the cleavage site “E³⁷³-³⁷⁴A” refers to the ITEGE³⁷³-³⁷⁴ARGS bond of human aggrecan as well as to the homologousaggrecanase-sensitive cleavage site in aggrecan from various animalspecies, the cleavage site “E¹⁵⁴⁵-¹⁵⁴⁶G” refers to the SELE¹⁵⁴⁵-¹⁵⁴⁶GRGTbond of human aggrecan as well as to the homologousaggrecanase-sensitive cleavage site in aggrecan from various animalspecies, the cleavage site “E¹⁷¹⁴-¹⁷¹⁵G” refers to the KEEE¹⁷¹⁴-¹⁷¹⁵GLGSbond of human aggrecan as well as to the homologousaggrecanase-sensitive cleavage site in aggrecan from various animalspecies, the cleavage site “E¹⁸¹⁹-¹⁸²⁰A” refers to the TAQE¹⁸¹⁹-¹⁸²⁰AGEGbond of human aggrecan as well as to the homologousaggrecanase-sensitive cleavage site in aggrecan from various animalspecies, the cleavage site “E¹⁹¹⁹-¹⁹²⁰L” refers to the ISQE¹⁹¹⁹-¹⁹²⁰LGQRbond of human aggrecan as well as to the homologousaggrecanase-sensitive cleavage site in aggrecan from various animalspecies.

The term “aggrecan” as used herein refers to the aggregatingproteoglycan, aggrecan, of cartilage from human or various animalspecies, as the native aggrecan isolated from tissue, as recombinantfull-length aggrecan or as a recombinant protein representing a portionof the aggrecan molecule.

As used herein the term “ADMP-susceptible cleavage site” refers to theE³⁷³-³⁷⁴A bond, the E¹⁵⁴⁵-¹⁵⁴⁶G bond, the E¹⁵⁴⁵-¹⁵⁴⁶G bond, theE¹⁸¹⁹-¹⁸²⁰A bond, and the E¹⁹¹⁹-¹⁹²⁰L bond of aggrecan from human andvarious animal species, and to a peptide bond of a protein containing anamino acid sequence which has a glutamine in the P1 position and showsat least 65% homology with the P1, P2, P3, P1′, P2′ and P3′ amino acidsof one or more of the ADMP-sensitive sites in the aggrecan molecule.

The term “sissel bonds” refers to the peptide bond of a polypeptide thatis to be cleaved by a protease. The term “P1” as used herein refers tothe amino acid residue on the N-terminal side of the sissel bond. Theterm “P2” as used herein refers to the amino acid residue adjacent to P1on the N-terminal side of the sissel bond. The term “P3” as used hereinrefers to the amino acid residue adjacent to P2 on the N-terminal sideof the sissel bond. The term “P 1′” as used herein refers to the aminoacid residue on the C-terminal side of the sissel bond. The term “P2′”as used herein refers to the amino acid residue adjacent to P1′ on theC-terminal side of the sissel bond. The term “P3′” as used herein refersto the amino acid residue adjacent to P2′ on the C-terminal side of thesissel bond.

The term “BC-3 antibody” refers to a monoclonal antibody that reactsspecifically with the newly-formed amino-terminal sequence ARGS onfragments produced by proteolytic cleavage at the Glu³⁷³-Ala³⁷⁴ aggrecancleavage site, but does not recognize this same sequence of amino acidswhen they are present within the intact interglobular domain of theprotein. Hughes, C. E., et al., Biochem. J. 306:799-804, 1995.

The term “SEQ ID NO:1” refers to the peptide sequence:QTVTWPDMELPLPRNITEGE-ARGSVILTVKPIFEVSPSPL.

The term “SEQ ID NO:2” refers to the peptide sequence:QTVTWPDVELPLPRNITEGE-ARGSVILTAKPDFEVSPTAPE.

The term “SEQ ID NO:3” refers to the peptide sequence:ITFVDTSLVEVTPTTFKEEE-GL-GSVELSGLPSGELGVSGT.

The term “41-PS” and “SEQ ID NO:4” refer to the peptide sequence:QTVTWPDMELPLPRNITEGEARGSVILTVKPIFEVSPSPL- (BIOTINYL)K.

The term “SEQ ID NO:5” refers to the peptide sequence:ARGSVILTVKPIFEVSPSPL-(BIOTINYL)K.

The term “SEQ ID NO:6” refers to the peptide sequence: K(BIOTINYL)-QTVTWPDMELPLPRNITEGE.

The term “30-IP” and “SEQ ID NO:7” refer to the peptide sequenceQTVTWPDMELPLPRNITEGQARGSVILTV--(BIOTINYL)K.

The invention can be further understood by the following examples. Theseexamples provide an illustration of embodiments of the invention andshould not be construed to limit the scope of the invention recited inthe appended claims. In the following examples all methods described areconventional unless otherwise specified.

EXAMPLE 1

Microplate Assay Format for Detection of ADMP Activity

The substrate and product peptides were prepared in the followingmanner. A 41 amino acid form (41-PS) SEQ ID NO:4 of the peptidesubstrate SEQ ID NO:1 was prepared by solid phase peptide synthesis. Thepeptide was prepared commercially (Quality Controlled Biochemicals, Inc.Hopkinton, Mass.) as a biotin conjugate by adding an additional lysineresidue at the carboxy terminus of the peptide SEQ ID NO: 1. Biotin wasthen covalently attached through the lysine ε-amino side chain. 41-PSsequence: (SEQ ID NO:4) QTVTWPDMELPLPRNITEGE-ARGSVILTVKPIFEVSPSPL-(BIOTINYL)K

A 21 amino acid peptide representing the product of ADMP-mediatedcleavage of the 41 -PS containing the ARGS N-terminus was prepared in asimilar manner and had the following sequence: (SEQ ID NO:5)ARGSVILTVKPIFEVSPSPL-(BIOTINYL)K

The substrate and product peptide microplates were prepared in thefollowing manner. A 0.1 mM stock of 41-PS was made by dissolving it indistilled water. From this a working solution of 7×10⁸ M 41-PS wasprepared in 1×PBS, 0.05% Tween 20. Aliquots of 100 μL of this solutionwere added to the microplate wells of a streptavidin coated microtiterstrip plate (DUPONT, NEN Products, Catalog# NEF-711). The solutions wereallowed to sit at 25° C. overnight in order for the biotin moiety of thepeptides to bind to the streptavidin which was coated on the microplate.The plate was washed three times with 200 μL of 1×PBS, 0.05% Tween 20,after which the plate was inverted, blotted dry, sealed and stored at 4°C.

The assay was performed in the following manner. Microplate strips(eight wells each) were rinsed once with 100 μL of 1×Assay Buffer (AssayBuffer consists of: 50 mM Tris, pH 7.5, 10 mM CaCl₂, and 100 mM NaCl)and blotted dry. Reactions were prepared in duplicate in a final volumeof 100 μL, containing: 50 μL of 2×Assay Buffer (100 mM Tris, pH 7.5, 20mM CaCl₂, and 200 mM NaCl), 25 μL of a hydroxymate inhibitor compound(final concentrations consisting of 5.0, 1.0, 0.75, 0.5, 0.25, 0.125,0.05, 0.001, and 0.0 μM), and 25 μL of soluble ADMP (0.05 ηM). Themicroplate strips were incubated for 3 hours at 37° C. The microplatewells were then washed six times with 200 μL of 1×PBS, 0.05% Tween 20using a Denley Well Wash 4 micro plate washer.

A BC-3 antibody solution was prepared by adding 4 μL of BC-3 antibody(0.405 mg/mL in PBS) to 2 mL of antibody dilution buffer (DB), whichconsisted of: 0.1 g BSA (Boehringer Mannheim Catalog 238-031), 10 mL1×PBS, and 10 μL Tween 20. 100 μL of this solution was added to eachwell. The microplate strips were incubated for 1 hour at 25° C. Themicroplate wells were washed six times with 200 μL of 1×PBS, 0.05% Tween20.

The secondary (detection) antibody solution was prepared by adding 4 μLof Goat anti-Mouse-HRP antibody conjugate (Pierce Cat #31430) (0.8 mg/mLin PBS) to 2 mL of antibody dilution buffer (DB). 100 μL of thissolution was added to each well. The microplate strips were incubatedfor 1 hour at 25° C. The microplate wells were washed six times with 200μL of 1×PBS, 0.05% Tween 20. TMB Substrate (100 μL per well, DAKO CatS1600) was added and the microplate strips incubated at 25° C. for 15minutes. The reaction was quenched with 100 μL of 1N HCl. The opticaldensity was read at 450 nm using a Molecular Devices Spectromax 250microplate reader. By coating the plate with the product peptide, onecan create a standard curve which can be used to convert optical densityvalues to units of activity.

ADMP activity can easily be followed by this method and inhibition ofADMP activity can be monitored. The IC₅₀ for the inhibition of ADMP bythe hydroxamate inhibitor tested was 0.413 μM.

EXAMPLE 2

HPLC Assay for Detection of ADMP Activity

The High Performance Liquid Chromatography (HPLC) instrument used forthe assay was from Hewlett-Packard, model number HP1090, equipped with aHP ChemStation. A 250.times.4.6 mm Vydac C₁₈ column with 10μ particlesize was obtained from The Separations Group, Tesperia, Calif.; HEPESfrom Research Organics, Cleveland, Ohio.; Brij-35 from Technicon Corp.,Tarrington, N.Y.; Other chemicals from Sigma, St. Louis, Mo.; thelyophilized peptide substrate 41-PS,QTVTWPDMELPLRNITEGEARGSVILT-VKPIFEVSPSPL-(BIOTINYL)K (SEQ ID NO:4),which includes the ADMP E373-A374 cleavage site, was obtained fromQuality Controlled Biochemicals, Inc. Hopkinton, Mass. A 21 amino acidproduct peptide (SEQ ID NO:5) with the sequence,ARGSVILTVKPIFEVSPSPL-(BIOTINYL)K, used as a standard for quantitation ofproduct formation, was obtained from the same vendor.

The HPLC assay is performed in the following manner. The reaction buffercontains 50 mM HEPES, 10 mM CaCl₂, 100 mM NaCl and 0.05% Brij-35, pH7.5. 30 μM 41-PS was incubated with 1 unit ADMP activity (1 unit=theamount of ADMP activity resulting in 1 pMole of 21-mer peptide productproduced per hr at 37°.) at 37° C. for 2.5 hours and then the reactionwas quenched with 50 mM EDTA. A 10 μL portion of the reaction mixturewas injected onto a reverse-phase HPLC C₁₈ column. The peptides wereeluted with a mobile phase of 0.1% trifluoroacetic acid and a 25-45%acetonitrile gradient in 20 minutes. UV absorbance was measured at 220nm and peak integration was performed on a Hewlett-Packard HPChemStation. The 21-mer product peptide was used as a standard forquantitation of product formation. The 41-PS and 21-mer product are wellseparated with retention times of 14.2 and 10.5 minutes, respectively. Astandard curve was prepared using the 21-mer peptide to allowquantitation of product formation. Effect of incubation time wasevaluated and found to be linear over the timecourse of the assay (FIG.2).

EXAMPLE 3

Inhibition of ADMP Activity by a Peptide Inhibitor

A peptide inhibitor was prepared based upon the sequence of the 40 aminoacid peptide SEQ ID NO: 1, but designed such that it contained a Glu toGln substitution at the P1 of the Glu373-Ala374 bond. This peptide(30-IP) of the sequence: (SEQ ID NO:7)QTVTWPDMELPLPRNITEGQARGSVILTVK-Biotin

was prepared with the n-terminus acetylated and the c-terminal residuepresent as the amide (Quality Controlled Biochemicals, Inc., Hopkinton,Mass.). This peptide was employed in a microplate assay as described inExample 1 wherein the 30-IP inhibitor was substituted for thehydroxamate inhibitor used in that example. The 30-IP inhibitor wasemployed at final concentrations of 0.01, 0.1, 1.0, 3.0, 5.0, 10.0, 30.0and 100.0 μM.

ADMP activity was inhibited as shown in FIG. 3. The IC₅₀ for theinhibition of ADMP activity by 30-IP was 11 μM.

EXAMPLE 4

Problot Assay

This example describes a method for analyzing ADMP enzymatic activityand inhibitors of this activity by monitoring cleavage at the E373-A374bond using the BC-3 antibody to detect fragments with the newN-terminus, ARGS. Samples containing ADMP activity (10 units/ml) wereincubated with 500 nM bovine aggrecan monomer in a final volume of 200μL in 0.05 M Tris, pH 7.6, containing 0.1 M NaCl and 10 mM CaCl₂.Reactions were incubated for 4 hr at 37° C., quenched with 20 mM EDTA,and analyzed for aggrecan fragments with the new N-terminus, ARGS,generated by specific ADMP-mediated cleavage using the Problot assay.

The Immobolin PVDF membrane plate (#MAIPN4550; Millipore Corp., Bedford,Mass.) was prewet with 50 μL per well 70% ethanol, incubated for 30seconds at room temperature then flushed two times each with 200 μL ofpurified H₂O. The plate was then coated with aggrecan equivalent to 36μg of glycosaminoglycan (GAG) as detected by the dimethyl methylene bluedye assay (Farndale R. W., et al., (1982) Conn. Tiss. Res. 9, 247-248)in 150 μL of 50 mM carbonate-bicarbonate buffer, pH 9.6, overnight at 4°C. with gentle agitation. The coating solution was then filtered off ofthe plate using a vacuum manifold and membranes were washed once with200 μL of Buffer A (Buffer A comprises 20 mM Tris, 500 mM NaCl, pH 7.5)allowing 20 seconds of contact with the membrane. Membranes were thenblocked with 150 μL of 5% BSA/TBS solution for 1 hour at roomtemperature with gentle agitation. The blocking solution was filteredoff of the plate and the membranes washed one time with 200 μL of 1×TBSbuffer, allowing 20 seconds of contact with membrane per wash.

Removal of the glycosaminoglycan (GAG) side chains from aggrecan isnecessary for the BC-3 antibody to recognize the epitope on the coreprotein. Therefore, to remove GAGs from the bound aggrecan, samples weretreated with deglycosylation enzymes as follows: 0.01 unitschondroitinase ABC (#EC4.2.2.4; Seikaguku Co., Kogyo, Japan) per 10 μgGAG in 150 μL of Buffer B (Buffer B comprises 50 mM sodium acetate, 100mM NaCl, pH 6.5) was added to each well and incubated at 37° C. for 1hour. Following incubation the enzyme solution was filtered out of theplate and 0.01 units chondroitinase ABC per 10. μg GAG, 0.01 unitskeratanase I (#EC3.2.1.103; Co., Kogyo, Japan) per 10 μg GAG, and 0.0005units keratanase II (Seikaguku Co., Kogyo, Japan) per 10 μg GAG in 150μL Buffer B were added and allowed to incubate an additional 2 hours at37° C. Enzyme solution was filtered out and membranes rinsed one timewith 200 μL of Buffer A.

150 μL of BC-3 antibody was added at a 1:500 dilution in 1% BSA inBuffer A and incubated for 1 hour at room temperature with gentleagitation. BC-3 antibody was removed and membranes washed three timeseach with 200 μL Buffer A allowing membrane contact for 20 seconds perwash. Next 150 μL of goat anti-mouse IgG AP conjugate (#S3721; Promega,Madison, Wis.) was added at a 1:2500 dilution in 1% BSA/TBS buffer andallowed to incubate for 1 hour at room temperature with gentleagitation. The secondary antibody was prefiltered prior to use, using0.22 μM syringe filters to remove aggregates which cause highbackground. Following the incubation, secondary antibody was removed andwells were washed three times each with 200 μL Buffer A allowing contactfor 20 seconds per wash. Then 100 μL of p-NPP AP substrate solution(#50-80-00; Kirkegaard & Perry Lab., Gaithersburg, Md.) was added toeach well and incubated in the dark at room temperature for 30 minutes.The solution was then filtered into a corresponding ELISA plate. Thefilter plate was washed with 100 μL of 500 mM EDTA and the wash wascombined with the corresponding samples in the ELISA plate. Absorbanceof the samples was read at 405 nm (Thermomax plate reader).

By coating the plate with the target peptide, ARGS, linked to BSArepresenting the ADMP-generated product, one can create a standard curvewhich can be used to convert optical density values to units ofactivity. A unit of ADMP activity is defined as that resulting inproduct produced equivalent to 1 μg BSA peptide per hour at 37° C.

To evaluate inhibition of ADMP activity, compounds are prepared as 10 mMstocks in dimethyl sulfoxide (DMSO), water or other solvents and dilutedto appropriate concentrations in water. Drug (50 μL) was added to 50 μLof 2 mg/mL aggrecan substrate and 50 μl of ADMP (40 units/ml) andbrought to a final volume of 200 μL by addition of 50 μl of 0.2 M Tris,pH 7.6, containing 0.4 M NaCl and 40 mM CaCl₂. The reaction mixture wasincubated for 4 hr at 37° C., quenched with 20 mM EDTA and analyzed forADMP-generated products. A sample containing enzyme and substratewithout drug was included as a positive control and enzyme prequenchedwith EDTA served as a measure of background.

IC₅₀ values for inhibitors of ADMP enzymatic activity determined usingthe Problot assay with the BC-3 antibody for analysis of productgeneration by cleavage at the E373-A374 bond correlated (r²=0.99) withthose determined using a BC-3 Western blot analysis to detect productformation.

1. A method for the determination of the presence of aggrecan-degradingmetalloprotease activity comprising: (a) binding an ADMP substratepeptide to a streptavidin-coated microtiter plate; (b) rinsing themicrotiter plate with assay buffer; (c) incubating the microtiter platewith an ADMP-containing sample; (d) rinsing the microtiter plate; (e)incubating the microtiter plate with a neoepitope antibody solution; (f)rinsing the microtiter plate; (g) incubating microtiter plates withsecondary-detection antibody solution; (h) incubating the microtiterplate with an appropriate substrate solution; (i) quenching thereaction; and (j) reading the optical density.
 2. The method of claim 1,wherein said ADMP peptide substrate comprises a covalently-linkedlinking-moiety.
 3. A method for the determination of ADMP activity byquantifying the appearance of a product peptide comprising: (a)incubating an ADMP substrate peptide with assay buffer and anADMP-containing sample; (b) quenching the reaction; (c) injecting aportion of the reaction mixture onto a reverse-phase HPLC column; (d)eluting the peptide with an organic solvent; (e) reading the absorbance;and (f) determining the quantity based on a standard curve.
 4. A methodfor assaying compounds for activity against an ADMP comprising: (a)providing an ADMP and an ADMP substrate; (b) contacting said ADMP with acandidate inhibitory compound in the presence of said ADMP; and (c)measuring the inhibition of the ADMP activity.
 5. An assay for detectingADMP activity which comprises: (a) incubating a sample containingsoluble ADMPs or aggrecanase activity with an aggrecan substrate; and(b) monitoring production of aggrecan fragments produced by specificcleavage at an ADMP-susceptible site using a neoepitope antibody to thenew N-terminus or the new C-terminus generated by specific ADMP-mediatedcleavage by the Problot assay comprising: (1) incubating apolyvinyl-denedifluoride (PVDF) cationically charged membrane, securedin a welled filtration plate, with a sample containing ADMP-degradedaggrecan; (2) washing any unbound aggrecan from the filtration plate;(3) coupling any unreacted cationic sites on the PVDF membrane with asolution of bovine serum albumin (BSA); (4) washing any unbound BSA fromthe filtration plate; (5) removing glycosaminoglycan side chains fromthe bound aggrecan with deglycosylation enzymes, wash membrane; (6)incubating PVDF membrane with a neoepitope antibody to fragmentsgenerated by cleavage at an ADMP-sensitive site, wash membrane; (7)incubating PVDF membrane with secondary detection antibody, washmembrane; (8) incubating PVDF membrane with detection substrate; and (9)draining solution into welled plate, obtain absorbance readings onindividual samples; compare values to those obtained for standard curve.6. An assay according to claim 5 wherein the sample is derived fromcartilage or chondrocytes.
 7. An assay according to claim 5 wherein theaggrecan substrate is native aggrecan isolated from human or animaltissue.
 8. An assay according to 5 wherein the aggrecan substrate is arecombinant aggrecan molecule or recombinant portion of the aggrecanmolecule containing an aggrecanase-sensitive cleavage site.
 9. An assayaccording to claim 5 wherein the recombinant portion of the aggrecanmolecule contains the E³⁷⁴-³⁷⁴A bond.
 10. An assay according to claim 5wherein the recombinant aggrecan fragment contains the EL¹⁵⁴⁵-⁵⁴⁶G bond.11. An assay according to claim 5 wherein the portion of the aggrecanmolecule contains the E¹⁷¹⁴-¹⁷¹⁵G bond.
 12. An assay according to claim5 wherein the recombinant portion of the aggrecan molecule contains theE¹⁸¹⁹-¹⁸²⁰A bond.
 13. An assay according to claim 5 wherein therecombinant portion of the aggrecan molecule contains the E¹⁹¹⁹-¹⁹²⁰Lbond.
 14. A method according to claim 5 wherein the neoepitope antibodyrecognizes the new N-terminus or new C-terminus generated by cleavage atthe E³⁷³ -A³⁷⁴ bond.
 15. The method of claim 5 wherein the neoepitopeantibody is a BC-3 monoclonal antibody.
 16. The method of claim 5wherein the neoepitope antibody recognizes the new N-terminus or newC-terminus generated by cleavage at the E¹⁵⁴⁵-G¹⁵⁴⁶ bond.
 17. The methodof claim 5 wherein the neoepitope antibody recognizes the new N-terminusor new C-terminus generated by cleavage at the E¹⁷¹⁴-G¹⁷¹⁵ bond.
 18. Themethod of claim 5 wherein the neoepitope antibody recognizes the newN-terminus or new C-terminus generated by cleavage at the E¹⁸¹⁹-A¹⁸²⁰bond.
 19. The method of claim 5 wherein the neoepitope antibodyrecognizes the new N-terminus or new C-terminus generated by cleavage atthe E¹⁹ ⁹-L¹⁹²⁰ bond.